Investigation on mechanism for expansion of GM-CSF autoantibody by next generation sequencing.

• Project/Area Number: 24390208
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Respiratory organ internal medicine
• Research Institution: Niigata University
• Principal Investigator: NAKATA Koh 新潟大学, 医歯学総合病院, 教授 (80207802)
• Co-Investigator(Kenkyū-buntansha): TAZAWA Ryushi 新潟大学, 医歯学総合病院, 准教授 (70301041) ; NAKAGAKI Kazuhide 日本獣医生命科学大学, 獣医学部, 准教授 (90143635) ; INOUE Yoshikazu 近畿中央胸部疾患センター, 臨床研究センター, センター長 (90240895) ; NEI Takahito 日本医科大学, 医学部, 助教 (30597670) ; TAKIZAWA Jun 新潟大学, 医歯学総合病院, 准教授 (70463990) ; YOKOSAKI Yasuyuki 広島大学, 保健センター, 准教授 (80210607)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥15,600,000 (Direct Cost: ¥12,000,000、Indirect Cost: ¥3,600,000); Fiscal Year 2014: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2013: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2012: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
• Keywords: 肺胞蛋白症 / 自己免疫疾患 / 肺胞マクロファージ / 自己抗体 / 次世代シークエンス / バイオインフォーマティクス / クラススイッチ / 体細胞超変異 / 抗GM-CSF自己抗体 / 抗体H鎖可変部

Outline of Final Research Achievements

It remains unclear why GM-CSF autoantibody is excessively produced in the patients. By preliminary evaluation using blood lymphocytes obtained from the patients and healthy subjects, we found high affinity IgG type GM-CSF autoantibody producing B cells expanded only in the patients but not in the healthy subjects, although IgM type producing B cells were even between them. It is suggested that class switching and clonal expansion are proceeding in the peripheral lymphnodes in the patients. To elucidate this, we tried to analyze the amino acid sequences of heavy chain variable regions using the next generation sequencing and a bioinformatics tool. Our analysis revealed that CDR1,2,and 3 amino acid sequences were variable and even in IgM clones but some specific sequences were exclusively used in IgG-type clones, suggesting oligo clonal expansion of IgG-type GM-CSF autoantibody clones in the patients.

Research Products

• [Journal Article] Novel aspects on the pathogenesis of Mycoplasma pneumoniae pneumonia and therapeutic implications. (2014)
  • Author(s): Saraya T, Kurai D, Nakagaki K, Sasaki Y, Niwa S, Tsukagoshi H, Nunokawa H, Ohkuma K, Tsujimoto N, Hirao S, Wada H, Ishii H, Nakata K, Kimura H, Kozawa K, Takizawa H, Goto H.
  • Journal Title: Front Microbiol Volume: 5 Pages: 410-410
  • DOI: 10.3389/fmicb.2014.00410
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Prenatal molecular diagnosis of X-linked hydrocephalus via a silent C924T mutation in the L1CAM gene (2014)
  • Author(s): Serikawa T, Nishiyama K, Tohyama J, Tazawa R, Goto K, Kuriyama Y, Haino K, Kanemura Y, Yamasaki M, Nakata K, Takakuwa K, Enomoto T.
  • Journal Title: Congenit Anom (Kyoto) Volume: 54 Issue: 4 Pages: 243-245
  • DOI: 10.1111/cga.12069
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Up-regulation of cluster of differentiation (CD) 11b expression on the surface of canine granulocytes with human granulocyte-macrophage colony-stimulating factor (GM-CSF). (2014)
  • Author(s): Nakagaki K, Nunomura Y, Uchida K, Nakata K, Tazawa R.
  • Journal Title: J Vet Med Sci Volume: 76(8) Pages: 1173-6
  • NAID: 130004432194
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Secondary pulmonary alveolar proteinosis complicating myelodysplastic syndrome results in worsening of prognosis: a retrospective cohort study in Japan (2014)
  • Author(s): Haruyuki Ishii, John F Seymour, Ryushi Tazawa, Yoshikazu Inoue, Naoyuki Uchida, Aya Nishida, Yoshihito Kogure, Takeshi Saraya, Keisuke Tomii, Toshinori Takada, Yuko Itoh, Masayuki Hojo, Toshio Ichiwata, Hajime Goto, Koh Nakata
  • Journal Title: BMC Pulmonary Medicine Volume: 14: 37 Issue: 1 Pages: 1471-2466
  • DOI: 10.1186/1471-2466-14-37
  • Peer Reviewed / Open Access
• [Journal Article] Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments long-term bioactivity with delayed clearance in vitro (2014)
  • Author(s): Hashimoto A, Tanaka T, Itoh Y, Yamagata A, Kitamura N, Tazawa R, Nakagaki K, Nakata K.
  • Journal Title: Cytokine Volume: 68 Issue: 2 Pages: 118-126
  • DOI: 10.1016/j.cyto.2014.03.009
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Standardized serum GM-CSF autoantibody testing for the routine clinical diagnosis of autoimmune pulmonary alveolar proteinosis (2014)
  • Author(s): Uchida K, Nakata K, Carey B, Chalk C, Suzuki T, Sakagami T, Koch DE, Stevens C, Inoue Y, Yamada Y, Trapnell BC.
  • Journal Title: J Immunol Methods Volume: 402 Issue: 1-2 Pages: 57-70
  • DOI: 10.1016/j.jim.2013.11.011
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] CYFRA 21-1 as a disease severity marker for autoimmune pulmonary alveolar proteinosis. (2014)
  • Author(s): Arai T, Inoue Y, Sugimoto C, Inoue Y, Nakao K, Takeuchi N, Matsumuro A, Hirose M, Nakata K, Hayashi S.
  • Journal Title: Respirology Volume: 19 Issue: 2 Pages: 246-252
  • DOI: 10.1111/resp.12210
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Duration of benefit in patients with autoimmune pulmonary alveolar proteinosis after inhaled granulocyte-macrophage colony-stimulating factor therapy. (2014)
  • Author(s): Tazawa R, Inoue Y, Arai T, Takada T, Kasahara Y, Hojo M, Ohkouchi S, Tsuchihashi Y, Yokoba M, Eda R, Nakayama H, Ishii H, Nei T, Morimoto K, Nasuhara Y, Ebina M, Akira M, Ichiwata T, Tatsumi K, Yamaguchi E, Nakata K.
  • Journal Title: Chest Volume: 145 Issue: 4 Pages: 729-737
  • DOI: 10.1378/chest.13-0603
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Clinical features of three cases with pulmonary alveolar proteinosis secondary to myelodysplastic syndrome developed during the course of Behçet's disease. (2013)
  • Author(s): Handa T, Nakatsue T, Baba M, Takada T, Nakata K, Ishii H
  • Journal Title: Respiratory Investigation Volume: 52 Issue: 1 Pages: 75-79
  • DOI: 10.1016/j.resinv.2013.05.005
  • Peer Reviewed / Open Access
• [Journal Article] Light chain (κ/λ) ratio of GM-CSF autoantibodies is associated with disease severity in autoimmune pulmonary alveolar proteinosis. (2013)
  • Author(s): Nei T, Urano S, Itoh Y, Kitamura N, Hashimoto A, Tanaka T, Motoi N, Kaneko C, Tazawa R, Nakagaki K, Arai T, Inoue Y, Nakata K.
  • Journal Title: Clin Immunol Volume: 149(3) Issue: 3 Pages: 357-364
  • DOI: 10.1016/j.clim.2013.10.002
  • Peer Reviewed
• [Journal Article] Serum VEGF-D concentration as a biomarker of lymphangioleiomyomatosis severity and treatment response:a prospective analysis of the Multicenter International Lymphangioleiomyomatosis Efficacy of Sirolimus (MILES) trial. (2013)
  • Author(s): Young L, Lee HS, Inoue Y, Moss J, Singer LG, Strange C, Nakata K, Barker AF, Chapman JT, Brantly ML, Stocks JM, Brown KK, Lynch JP 3rd, Goldberg HJ, Downey GP, Swigris JJ, Taveira-DaSilva AM, Krischer JP, Trapnell BC, McCormack FX; MILES Trial Group.
  • Journal Title: The Lancet Respiratory Medicine. Volume: 1(6) Issue: 6 Pages: 445-452
  • DOI: 10.1016/s2213-2600(13)70090-0
  • Peer Reviewed
• [Journal Article] Mycoplasma pneumoniae extract induces an IL-17-associated inflammatory reaction in murine lung: Implication for mycoplasmal pneumonia. (2013)
  • Author(s): Kurai D, Nakagaki K, Wada H, Saraya T, Kamiya S, Fujioka Y, Nakata K,Takizawa H, Goto H
  • Journal Title: Inflammation Volume: 36(2) Issue: 2 Pages: 285-293
  • DOI: 10.1007/s10753-012-9545-3
  • Peer Reviewed
• [Journal Article] 肺胞蛋白症に見られる易感染性 (2013)
  • Author(s): 赤坂圭一、中田光
  • Journal Title: 化学療法の領域 Volume: 29(12) Pages: 28-35
  • Peer Reviewed
• [Journal Article] 肺胞蛋白症とGM-CSF自己抗体 (2013)
  • Author(s): 中田光
  • Journal Title: 検査と技術 Volume: 41(13) Pages: 1224-1229
  • Peer Reviewed
• [Journal Article] 自己培養骨膜シートを用いた歯槽骨再生研究-データをどのように評価するか- (2013)
  • Author(s): 奥田一博、吉江弘正、川瀬知之、 中田光
  • Journal Title: 新潟医学会雑誌（別刷） Volume: 127(7) Pages: 349-354
  • Peer Reviewed
• [Journal Article] IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis. (2012)
  • Author(s): Nei T, （10名略）Tazawa R, Nakata K.
  • Journal Title: Am J Physiol Lung Cell Mol Physiol. Volume: 302(9) Issue: 9 Pages: 1959-64
  • DOI: 10.1152/ajplung.00378.2011
  • Peer Reviewed
• [Journal Article] Runx1 deficiency in CD4+ T cells causes fatal autoimmune inflammatory lung disease due to spontaneous hyperactivation of cells. (2012)
  • Author(s): Wong WF, Kohu K, Nakamura A, Ebina M, Kikuchi T, Tazawa R, Tanaka K, Kon S, Funaki T, Sugahara-Tobinai A, Looi CY, Endo S, Funayama R, Kurokawa M, Habu S, Ishii N, Fukumoto M, Nakata K, Takai T, Satake M.
  • Journal Title: J Immunol. Volume: 188(11) Pages: 5408-5420
  • Peer Reviewed
• [Journal Article] Bilateral Peripheral Infiltrates Refractory to Immunosuppressants were Diagnosed as Autoimmune Pulmonary Alveolar Proteinosis and Improved by Inhalation of Granulocyte/Macrophage-Colony Stimulating Factor (2012)
  • Author(s): Satoh H, Tazawa R, （4名略）, Nakata K, Nukiwa T.
  • Journal Title: Internal Medicine Volume: 51 Issue: 13 Pages: 1737-1742
  • DOI: 10.2169/internalmedicine.51.6093
  • NAID: 130002062188
  • ISSN: 0918-2918, 1349-7235
  • Peer Reviewed
• [Journal Article] 肺胞蛋白症の十年史 (2012)
  • Author(s): 中田 光
  • Journal Title: 呼吸 Volume: 31(9) Pages: 845-848
  • Peer Reviewed
• [Presentation] Pulmonary Alveolar Proteinosis (PAP) And Inhaled Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) Therapy-Clinical Features Predicting Recurrence. (2012)
  • Author(s): R. Tazawa, T. Arai, T. Takada, Y. Kasahara, Y. Tsuchihashi, T. Nei, M. Hojo, H. Nakayama, M. Yokoba, H. Ishii, R. Eda, Y. Nasuhara, M. Ebina, M. Akira, E. Yamaguchi, Y. Inoue, K. Nakata.
  • Organizer: Meet The Professor Meeting.
  • Place of Presentation: ドイツ(招待講演)
  • Year and Date: 2012-06-23
• [Presentation] IgM Type GM-CSF Autoantibody is Etiologically a Bystander but Maybe Involved in Development of IgG Type Autoantibody in Autoimmune Pulmonary Alveolar Proteinosis. (2012)
  • Author(s): Nakata K, Nei T, Motoi N, Urano S, Tazawa R, Kanako C, Nakagaki K, Suzuki M, Takizawa J, Azuma A.
  • Organizer: American Thoracic Society Annual Meeting.
  • Place of Presentation: アメリカ
  • Year and Date: 2012-05-23
• [Presentation] Evidence for Permeation of IgG type GM-CSF Autoantibody from the Circulation to the Lung in Autoimmune Pulmonary Alveolar Proteinosis. (2012)
  • Author(s): Urano S, Akasaka K, Nei T, Motoi N, Tazawa R, Nakata K.
  • Organizer: American Thoracic Society Annual Meeting.
  • Place of Presentation: アメリカ
  • Year and Date: 2012-05-23
• [Presentation] Repertoire Analysis of GM-CSF Autoantibody mRNA by Next Generation Sequencing In Autoimmune Pulmonary Alveolar Proteinosis. (2012)
  • Author(s): Nakata K, Nei T, Motoi N, Urano S, Tazawa R, Nakagaki K, Suzuki M, Takizawa J, Azuma A.
  • Organizer: American Thoracic Society Annual Meeting.
  • Place of Presentation: アメリカ
  • Year and Date: 2012-05-23
• [Book] 大学病院などの再生医療を支える細胞プロセッシング室運営マニュアル (2012)
  • Author(s): 井上典子, 梶正美, 小神晴美, 渡辺真理, 関根優, 白山早起, 藤本陽子, 瀧澤淳, 牧口智夫, 布施一郎, 中田光
  • Total Pages: 142
  • Publisher: 株式会社ウイネット